Measuring protein Activity (Spectroscopy) Flashcards
Why do we measure proteins?
- identity
- concentration
- activity
What are some destructive ways of measuring protein conc?
- Bradford Assay
- BCA Assay (denatures proteins)
What is spectroscopy?
the study of how electromagnetic radiation interacts with matter
How does spectroscopy work?
- energy carried by electromagnetic radiation is dependent on the wavelength and frequency
- electromagnetic energy is absorbed by macromolecules
- Uses UV-visible light
What are the types of spectroscopy?
- NMR
- Infrared
- Ultraviolet-Visible
When you have a short wavelength do you have a high or low amount of energy?
high
What is the plank-einstein equation?
E= hc/ λ
c=f λ
E=hf
E=energy h=plank constant c= speed of light λ= wavelength f=frequency
How is absorbance measured?
the amount of energy it takes an electron to move from the highest occupied orbital to the lowest unoccupied orbital
How is absorbance used to measure protein activity?
Wavelength depends on the energy needed.
Biological molecules absorb varying wavelengths of light dependent on the structure and concentration of the molecule.
What residues absorb at a wavelength of 280nm?
Aromatic residues:
Phenylalanine
Tryptophan
Tyrosine
What happens to the absorbance when there are more aromatic rings present?
Absorbance increases
What wavelength does DNA absorb at?
260nm
What wavelength do peptide bonds and carboxylic moieties absorb at?
200-230nm
What equipment is used in spectroscopy?
Basic spectrophotometer:
- Light source is split in two wavelengths by monochromator
- Sample in cuvette absorbes wavelength
- The amount not absorbed is recorded.
How do you calculate absorbance?
A λ= log10(I0/I)
I0=incident energy
I=transmitted energy
What does the absorbance depend on?
- concentration of solute (c)
- light path length (l)
- molar absorption/extinction coefficient
What is the path length normally?
1cm
What is the Beer-Lambert equation?
A λ= ελcl
ελ= absorbance at 1M^-1cm^-1 solution at wavelength λ of incident energy
What is absorbance proportional to?
The concentration of the solute (C)
How do you calculate protein concentrations using spectroscopy?
- make a calibration curve using known concentrations
- measure absorbance of unknown compound and use curve to work out conc
How can you work out protein concs in an enzyme reaction?
-as reaction goes on, more product is formed and substrate is used up so couple this with absorbance
MUST CONSIDER STOCHIOMETRY BECAUSE SOMETIMES 2 MOLES OF PRODUCT ARE FORMED FROM 1 MOLE OF SUBSTRATE AND VICE VERSA
What happens if you can’t use spectroscopy to measure protein conc (no aromatic rings)
- link it to something you can see
e. g a co-factor that changes structure as enzyme works - link first reaction to another reaction that you can see the products of
Why is rate of reaction important when you use coupled reactions?
because if the rate of reaction for the second reaction is slower than the first reaction it won’t truly reflect the rate of reaction of the first reaction (second reaction needs to be going at the same or a faster rate)
How can you ensure that the second reaction is going quickly?
use excess enzyme
If there are other contaminants present in all of the reactions, how do you measure absorbance?
- add all the absorbances up and compare them fro each different stage
- if you know the amount of absorbance of the contaminants, you know the amount of absorbance fr your compound
Is it ok to have contaminants in your reaction?
yes as long as they stay the same
What must be considered when coupling reactions?
Visible signal difference
Reaction 2 is not rate limiting
Consider stoichiometry
